Bioethanol is being blended with gasoline 5-80% as transportation fuel in different countries to substantiate the rising price of crude oil and such high-octane blended fuel that reduces CO2 and NOx emission from the automobiles.
Lignocellulosic biomass based ethanol production has many advantages. The large scale availability of biomass at a cheaper cost, environmentally benign bioprocessing to produce ethanol, national energy security, competitive alcohol based downstream chemicals, macroeconomic benefits for rural communities and the society at a large, has accelerated the biomass based bioethanol production worldwide. The lignocellulosic feed stock does not compete for food and land available for food production. Bioethanol as fuel is carbon neutral when produced from lignocellulosic biomass.
Application of thermophilic microorganisms that are capable of producing ethanol at higher temperature is highly demanded in the industry to reduce the cost of ethanol purification. Further, important advantages of using thermophilic microorganisms i.e.; bacteria and yeast at higher temperature include high rate of sugar metabolism, tolerance to high salt and solvent concentration, saving of energy on cooling of fermentation process and recovery of ethanol and significant restriction of contamination chances. Application of thermophilic yeast for ethanol production from different mono and disaccharides and carbohydrates would have advantages over thermophilic bacteria because of higher genetic stability and higher metabolic capacity as compared to bacteria.
Reference may be made to (Ghosh P and Ghose T K, Advanced Biochemical Engineering/Biotechnology, Vol 20, Biotechnology in India II, Scheper T (ed.) Springer, New York, 1-27, 2003) wherein bioethanol is being produced from molasses in India or from sugarcane juice in Brazil or from cereal grains in USA and Europe by fermentation process using yeast, Saccharomyces cereviciae or bacteria Zymomonas mobilis and other microorganisms in the temperature range of 28-35° C. The draw backs are the ethanol present in the fermented broth at low concentration (6-9%) is further purified to 99.9% by distillation, rectification, azeotropic distillation and dehydration process at temperature range of 70-100° C. with high input of energy which is a major process cost for ethanol production. The Saccharomyces cereviciae or bacteria Zymomonas mobilis are not capable of utilizing different mono and disaccharides except glucose and sucrose to ethanol limiting the scope of utilization of these microorganisms for ethanol production from lignocellulosic and starch biomass. The optimum temperature of growth and fermentation limits to 32-37° C. causing contamination problem. The microorganisms, Saccharomyces cereviciae and Zymomonas mobilis have low tolerance to ethanol concentration up to 8%.
Reference may be made to (Romero et al., Biochemical Engineering Journal, 36, 108-115, 2007) wherein ethanol fermentation of olive tree pruning hydrolysate with Pachysolen tannophilus has been reported with an ethanol yield of 0.38 g/g sugar in hydrolysate at 30° C. and pH 3.5. The conversion of the hemicellulose fraction was 92% in around 300 h. The draw backs are the substrate consumption rate and ethanol production rate are very slow. The strain utilizes only pentose sugars to ethanol, no hexose sugars utilization reported.
Reference may be made to (Cheng et al., Biotechnology Letters, 29, 1051-1055, 2007) wherein sugarcane bagasse hemicellulose hydrolysates has been fermented to ethanol using Pachysolen tannophilus DW06 giving 21 g/l ethanol; the yield being 0.35 g/g sugar and the productivity of 0.59 g.l−1.h−1 at 30° C. The draw backs are the strain utilizes only pentose sugars to ethanol, no hexose sugars utilization reported. The ethanol productivity is low.
Reference may be made to (Katahira et al., Applied Microbiology and Biotechnology 72, 1136-1143, 2006) wherein a recombinant yeast strain Saccharomyces cerevisiae MT8-1 is constructed to ferment the xylose and cellooligosaccharides by integrating genes for the intercellular expressions of xylose reductase and xylitol dehydrogenase from Pichia stipitis, and xylulokinase from Saccharomyces cerevisiae and a gene for displaying β3-glucosidase from Aspergillus acleatus on the cell surface. In the fermentation of the sulfuric acid hydrolysate of wood chips, xylose and cellooligosaccharides are completely fermented after 36 h by the recombinant strain with 41% ethanol yield. The draw backs are the recombinant strain showed fermentation of xylose and oligosaccharides at temperature 32-35° C. requiring high energy input for the cooling the fermentation broth and recovery of ethanol by distillation. The specific productivity on hydrolysate was 0.4-0.42 g.g−1.h−1 in the batch process. The recombinant strains have the low stability.
Reference may be made to (Saha and Cotta, Biotechnology Progress, 22, 449-453, 2006) wherein an ethanol yield of 0.46 g/g of available sugars (0.29 g/g straw) using recombinant Escherichia coli strain FBR5 from the fermentation of alkaline peroxide pretreated and enzyme saccharified wheat straw hydrolysate is obtained at pH 6.5 and 37° C. in 48 h. The draw backs are the ethanol productivity 0.4 g.l−1.h−1 is low in batch process as compared to other prior art. The recombinant strains have the low stability.
Reference may be made to (U.S. Pat. No. 5,554,520 dated Oct. 9, 1996) wherein novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase have been transformed with genes coding for alcohol dehydrogenase and pyruvate. The recombinant strain E. coli TC4 has been constructed by transforming the alcohol dehydrogenase and pyruvate decarboxylase genes from Z. mobilis. The ethanol yield on glucose, lactose and xylose was 94%, 80% and 100% of theoretical yield respectively. The ethanol productivity is 0.64-1.4 g.l−1.h−1. The draw backs are the recombinant strain E. coli TC4 could ferment hexose and pentose sugars at 37° C. with low ethanol productivity. The recombinant strains have low stability.
Reference may be made to (U.S. Pat. No. 7,285,403 dated 23 Oct. 2007) wherein xylose-fermenting recombinant yeast strains expressing xylose reductase, xylitol dehydrogenase, and xylulokinase and having reduced expression of PHO13 or a PHO13 ortholog, used for fermenting xylose to obtain ethanol. The recombinant strain of S. cerevisiae is developed, by expressing XYL1 and XYL2 from P. stipitis under the control of the GAPHD (TDH1) promoter, a strong constitutive S. cerevisiae promoter, and engineered to contain multiple copies of XYL3 with its native P. stipitis promoter integrated into the S. cerevisiae genome using a tunable expression vector that allows various expression levels by achieving different integrated copy numbers. The maximum ethanol concentrations are 5.4 g/l and 10.7 g/l for the FPL-YSX3 and the FPL-YSX3P, respectively from 40 g/l of xylose. The draw backs are very low ethanol yield and fermentation occurred at 37° C.
Reference may be made to (Singh et. al; World Journal of Microbiology & Biotechnology 14, 823-834, 1998) wherein application of thermophilic yeast in ethanol production has been reported by using Kluyveromyces marxianus IMB-3 strain MTCC 1288 which showed maximum ethanol yield at 45° C. from various mono and disaccharide sugars. The yeast strain showed 35 gl−1 ethanol yields from 100 gl−1 sucrose in 13 hr in a batch fermentation process. Further application of yeast Kluyveromyces marxianus IMB-3 in an Indian distillery for industrial scale ethanol production from molasses showed 6-7.2% ethanol concentration in a batch fermentation of 18 hr as compared to Saccharomyces cerevisiae producing same concentration of ethanol in 25 hr. The drawbacks are production of ethanol from sucrose by thermophilic yeast such as Kluyveromyces marxianus IMB-3 strain MTCC 1288 could be achieved maximum at 45° C. with 68% of theoretical yield in batch fermentation. The highest productivity of ethanol from glucose and molasses by immobilized cells of Kluyveromyces marxianus IMB-3 could be achieved was 1.41 gl−1h−1 and 9.41 gl−1h−1 with ethanol yield of 90% to 68% of theoretical yield respectively in a continuous fermentation process. However, the drawback of the said process was that the strain was not used for ethanol production from lignocellulosic biomass hydrolysate and the strain showed lower productivity of ethanol on glucose and molasses that limits the scope of lowering of process cost.
Reference may be made to (U.S. Pat. No. 7,344,876 dated 18 Mar. 2008) wherein the yeast strains of the genus Kluyveromyces sp. are disclosed to produce ethanol from lignocellulosic waste materials. The strains have the capability to ferment cellulose, hexoses, pentoses, or hemicelluloses from waste materials to ethanol. The draw backs are the yeast Kluyveromyces sp. produced ethanol at very low yield and temperature 45° C. from lignocellulosics waste material.
Reference may be made to (Shin et. al; International Journal of Systematic and Evolutionary Microbiology, Vol 51, 2167-2170, 2001) wherein a thermophilic yeast strain, Candida thermophilia was isolated from soil of Korea that has maximum growth temperature of 50-51° C., along with certain other physiological characteristics. The drawback is the strain has very low ethanol yield.
Reference may be made to (U.S. Pat. No. 4,094,742) wherein a mixed culture of thermophilic cellulolytic sporocytophaga and thermophilic ethanol-producing Bacillus is admixed with a suspension of cellulose in nutrient mineral broth and the resulting mixture is fermented at a pH ranging from 7 to 8 and at a temperature of 50° C. to 65° C. to produce ethanol. The drawbacks are the mixed culture of fungus and bacteria that does not contain any yeast, could produce 10% ethanol based on cellulose used as substrate which is 40% of theoretical yield and slow rate of production @1.9 gl−1h−1.
Reference may be made to (Georgieva and Ahring, Applied Microbiology and Biotechnology, 77, 61-68, 2007) wherein the thermophilic anaerobic bacterial strain Thermoanaerobacter BGT1L1 has been used to ferment undetoxifed corn stover hydrolysate in a continuous immobilized reactor system at 70° C. with ethanol yield 0.39-0.42 g/g sugars consumed. Xylose in corn stover hydrolysate is utilized to produce ethanol by the bacteria nearly 89-98%. The draw backs are the sugar uptake and ethanol conc. in continuous process are very low.
Reference may be made to (WO/2007/053600 dated Oct. 5, 2007 and WO/2007/130984 dated 15 Nov. 2007) wherein mutant thermophilic organisms of Thermoanaerobacterium saccharolyticum that consume a variety of biomass derived substrates are disclosed. 30 g/l ethanol was produced in 140 hr when the SSF was performed with 80 g/l avicel. The draw backs are the ethanol productivity (0.214 gl−1h−1) is very low.
Reference may be made to (U.S. Pat. No. 5,182,199 dated 26 Jan. 1993) wherein the production of ethanol by a process in which a strain of Bacillus stearothermophilus or other thermophilic, facultative anaerobic bacterium at 70° C. is disclosed. The anaerobic fermentation is carried out with continuing removal of ethanol at 70° C. and the fermentative activity of the bacterium is maintained by withdrawing a proportion of the anaerobic fermentation medium on a continuing basis preferably with removal of ethanol and allowing the bacteria therein to multiply aerobically, using residual sugars or metabolites thereof present in the medium, before being returned to the anaerobic fermentation. The ethanol yield 91% of theoretical yield with productivity 0.6 gl−1h−1 is obtained with 5% w/v sucrose, where 97% sucrose is consumed. The draw backs are the bacteria utilized sucrose as raw material and showed low productivity on ethanol production. Above 4% w/v ethanol concentration ethanol inhibits the fermentation.
Reference may be made to (U.S. Pat. No. 5,580,389 dated Mar. 12, 1996 and U.S. Pat. No. 5,820,687 dated 13 Oct. 1998) wherein an economically viable method for producing sugars using concentrated acid hydrolysis of biomass containing cellulose and hemicellulose is disclosed. This method involves the use of a resin separation unit wherein the sugars are adsorbed on a strong acid resin. The resin separation unit is preferably a cross-linked polystyrene divinylbenzene cation exchange resin bed, wherein the resin is cross linked with divinylbenzene and treated with sulfuric acid to produce a strong acid resin. Preferably, the divinylbenzene is at a concentration of from about 6% to about 8%. The resin bed has a tapped bed density of 0.6 g/ml to 0.9 g/ml and the resin has a strong acid capacity of at least 2 meq/g. The resin used is DOW XFS 43281.01, available from Dow Chemical. At least 98% of the sugars in the hydrolysate are recovered on washing of the bed. Overall recovery of the acid is 97.3% and the recovery of the sugar is 95.5% at 60° C. The drawbacks are the recovery of sugars on washing will dilute the sugar conc. in the hydrolysate.
Reference may be made to (Appua Nestor et. al Society for Biosciences and Bioengineering Japan vol 60. p. 130, 1999) wherein reported is alcohol production by a thermophilic yeast Isolated from Philippine soil. The optimum growth of this yeast was observed at 37-40° C. and at pH 4.0-6.0 in a semi-synthetic medium. The drawbacks are the temperature range for ethanol fermentation is lower as compared to other prior art.
Accordingly, keeping in view the drawbacks of the hitherto known prior art, the inventors of the present invention realized that there exists a dire need to provide a novel strain of yeast that grows on sucrose, glucose, xylose, maltose, galactose, cellobiose in a wide temperature range and which ferments glucose, xylose, sucrose and starch to ethanol at high temperature in the range of 40-55° C. and at pH range of 3.5 to 5.5 showing maximum rate of ethanol production i.e., 13.8 gl−1h−1 on glucose in continuous fermentation process.